Microbial production of ectoine and hydroxyectoine as high-value chemicals

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a “bacterial milking” process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina. However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

Metabolic Engineering of Escherichia Coli for Ectoine Production With the Fermentation Strategy of Supplementing Amino Donor

Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.

Metabolic Engineering of Escherichia Coli for Ectoine Production with the Fermentation Strategy of Supplementing Amino Donor

Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.

Draft Genome Sequences of Vibrio alginolyticus Strain S6-61 and Vibrio diabolicus Strain S7-71, Isolated from Corals in the Andaman Sea

We report the draft genome sequences of Vibrio alginolyticus strain S6-61 and Vibrio diabolicus strain S7-71, isolated from the corals Pocillopora verrucosa and Fungia danai, respectively. The genomes of strains S6-61 and S7-71 contain 4,880 and 4,641 protein coding genes, respectively, and harbor genes associated with the ectoine biosynthesis pathway.

CosR is a repressor of compatible solute biosynthesis and transporter systems

Bacteria accumulate small, organic compounds, called compatible solutes, via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (ectABCasp_ect) and glycine betaine (betIBAproXWV), four betaine-carnitine-choline transporters (bcct1-bcct4) and a second ProU transporter (proVWX). Most of these systems are induced in high salt. CosR, a MarR-type regulator, which is divergently transcribed from bcct3, was previously shown to be a direct repressor of ectABCasp_ect in Vibrio species. In this study, we investigated the role of CosR in glycine betaine biosynthesis and compatible solute transporter gene regulation. Expression analyses demonstrated that betIBAproXWV, bcct1, bcct3, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant shows induced expression of these systems in the mutant at low salinity compared to wild-type. DNA binding assays demonstrate that purified CosR binds directly to the regulatory region of each system. In Escherichia coli GFP reporter assays, we demonstrate that CosR directly represses transcription of betIBAproXWV, bcct3, and proVWX. Similar to V. harveyi, we show betIBAproXWV is positively regulated by the LuxR homolog OpaR. Bioinformatics analysis demonstrates that CosR is widespread within the genus, present in over 50 species. In several species, the cosR homolog was clustered with the betIBAproXWV operon, which again suggests the importance of this regulator in glycine betaine biosynthesis. Incidentally, in four Aliivibrio species that contain ectoine biosynthesis genes, we identified another MarR-type regulator, ectR, clustered with these genes, which suggests the presence of a novel ectoine regulator. Homologs of EctR in this genomic context were present in A. fischeri, A. finisterrensis, A. sifiae and A. wodanis.ImportanceVibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems are repressed. However, the mechanism of this repression is not fully elucidated. CosR plays a major role in the repression of multiple compatible solute systems in V. parahaemolyticus as a direct negative regulator of ectoine and glycine betaine biosynthesis systems and four transporters. Homology analysis suggests that CosR functions in this manner in many other Vibrio species. In Aliivibrio species, we identified a new MarR family regulator EctR that clusters with the ectoine biosynthesis genes.

Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine

ABSTRACT To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus. In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR. Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness. IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus. We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.